Calcification is ubiquitous in vascular disease, and contributes significantly to morbidity and mortality. Matrix GLA protein (MGP) is an alleged calcification inhibitor that is up-regulated in atherosclerotic lesions. Deletion of the MGP gene results in arterial calcification and in morphological abnormalities in mice and humans. MGP is a secreted protein that is modified by vitamin K-dependent g-carboxylation. When isolated from bone, it is processed and lacks seven C-terminal amino acids. The mechanism of MGP is poorly understood. However, our experimental data points to a role in transforming growth factor-beta (TGF-beta) and bone morphogenetic protein (BMP) signaling. The data suggest a potential link between BMP and TGF-beta in that BMP2 and BMP-4 induce expression of the TGF-beta, receptor Activin-like Kinase receptor 1 (ALK1). Signaling through ALK1 induces MGP, which provides negative feedback regulation for BMP. Interactions between MGP and specific receptors were also observed. Three hypotheses will be tested in this grant. The first hypothesis is that MGP regulates TGF-beta and BMP signaling at the receptor level, resulting in altered SMAD signaling and gene expression. We will characterize activation of SMAD signaling and relevant reporter genes induced by TGF-beta, BMP-2 and BMP-4 in presence of MGP, and identify the TGF-beta / BMP receptors that are regulated by MGP. The second hypothesis is that MGP participates in specific protein-protein interactions that may be altered by C-terminal processing. We will characterize the interactions, which include the TGF-beta receptors, using specific immunoprecipitation in combination with a general proteomic approach. The third hypothesis is that MGP functions as a BMP inhibitor in developing arteries and in atherosclerotic lesions. We will generate a transgenic mouse for targeted, conditional expression of the BMP-inhibitor Noggin. Noggin expression will be induced in MGP null mice in attempt to replace MGP and abolish calcification. The effect of Noggin will also be determined in atherosclerotic lesions in LDL-receptor null mice. Our results will add insight to calcification of vessels and heart valves, a common problem in heart disease. In addition, it may provide information on how to design new treatments for unwanted calcification.